The functional importance of the fraction of the endoplasmic reticulum (ER) positioned closely to the plasma membrane (PM) has long been recognized, especially in phospholipids synthesis and -transfer in both metazoan cells and yeast. STIM1, a recently described ER protein rapidly translocates to such PM-adjacent ER compartment upon depletion of the ER calcium stores where it activates the newly identified calcium channel, Orai1, forming the molecular basis of the so-called store-operated calcium entry (SOCE) phenomenon. The importance of this calcium entry pathway is highlighted by the fact that mutations in Orai1 has been linked to severe inborn human immunodeficiencies and that this route of calcium entry is key to the calcium regulated activation of regulatory T-cells mediated by the NFAT transcription factors. Studying the molecular mechanism of STIM1 translocation to the PM-adjacent ER compartment and molecular definition of this compartment could aid identification of novel molecular targets for immunosupression. [unreadable] We used a chemically inducible molecular bridge formation between the PM and ER membranes to highlight the plasma membrane-adjacent ER compartment to show that this is the site where STIM1 and its calcium channel partner, Orai1 form a productive interaction upon ER calcium store depletion. By changing the length of the linkers connecting the plasma and ER membranes, we were able to show that Orai1 requires a larger space than STIM1 between the two membranes. This finding suggests that Orai1 is part of a larger macromolecular cluster with an estimated 11-14 nm protrusion to the cytoplasm, while the cytoplasmic domain of STIM1 fits in a space calculated to be less than 6 nm. We also showed that agonist-induced translocation of STIM1 is rapidly reversible and only partially affects STIM1 in the juxtanuclear ER compartment. These studies are the first to detect juxtaposed areas between the ER and the plasma membrane in living cells revealing novel details of STIM1-Orai1 interactions.[unreadable] [unreadable] A select group of plasma membrane receptors activate phospholipase C enzymes to hydrolyze the plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) and initiate a cytoplasmic Ca2+ increase to elicit a cellular response. The maintenance of the plasma membrane PIP2 pool is essential for proper cellular signaling and is provided by phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K) enzymes. We have previously identified two PI4Ks (type-III&#945; and &#946;) enzymes that are required for the sustained supply of PIP2 but could not determine which of the two was more important for this process. [unreadable] We used a combination of approaches to address this question. A large-scale screen of PI3K inhibitors revealed that one of these compounds, Pik93 showed significant potency against the PI4KIII&#946; but not the &#945; enzyme. We used molecular modeling based on the solved crystal structure of the PI3K&#947; enzyme and modelled the catalytic domains of both PI4Ks either with ATP or the inhibitors, wortmannin (Wm) and Pik93. These studies helped identify the structural differences that form the basis of the differential inhibitor sensitivities and allowed us to make mutations that changed the Wm sensitivity of the enzymes. In parallel studies we used protein modules fused to the green fluorescent protein (GFP) to visualize the cellular PI4P and PIP2 pools in living cells and showed that the synthesis of the plasma membrane PI4P and PIP2 pools can be blocked by inhibitors of the PI4KIII&#945; but not the PI4KIII&#946; enzyme. Finally, we used RNAi-mediated gene silencing to show that down-regulation of PI4KIIIa but not any of the other PI4K enzymes interfered with the supply of PI4P and PIP2 during agonist stimulation. These data suggested that PI4KIII&#945; is a critical enzyme for the production of the signaling pool of plasma membrane phosphoinositides. This finding is in good agreement with the conclusion of our parallel studies in zebrafish where the down-regulation of the fish ortholog of the enzyme caused developmental defects because of an impaired supply of PIP2 in the plasma membrane for PI3K signaling.[unreadable] [unreadable] Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. [unreadable] In collaboration with the Nathanson group c-Met was found to rapidly translocate to the nucleus upon stimulation with HGF. Intriguingly, the calcium signals that are induced by HGF result from PI(4,5)P2 hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus rather than within the cytoplasm. This conclusion was based on the observation that an InsP3 binding domain targeted to the nucleus was able to inhibit HGF but not vasopressin-induced Ca2+ signals, while the same domain expressed in the cytosol and excluded form the nucleus had a reverse effect, being inhibitory on vasopressin- but not HGF-induced Ca2+ signaling. Therefore, while vasopressin generated InsP3 at the plasma membrane, C-Met receptor stimulation appears to bypass the cytoplasm and generates the messenger in the nucleus. Translocation of c-Met to the nucleus was found dependent upon the adaptor protein Gab1 and importin 1, and formation of Ca2+ signals in turn depends upon this translocation. These studies suggest a unique mechanism of Ca2+ signaling that is well suited to affect primarily nuclear processes.